Selenocysteine inserting RNA elements modulate GTP hydrolysis of elongation factor SelB.
Elongation factor SelB is required for the incorporation of the amino acid selenocysteine into proteins in Escherichia coli. Selenocysteine incorporation is thought to be achieved by simultaneous binding of SelB to selenocysteyl-tRNASec and to an mRNA hairpin structure located 3' adjacent to the UGA selenocysteine codon. SelB was shown previously to bind to GTP or GDP in a molar ratio of 1:1. Here, we demonstrate that SelB, like EF-Tu, exhibits a low intrinsic GTPase activity in the absence of ribosomes. As shown for EF-Tu, GTPase activity of SelB is stimulated by the presence of E. coli 70S ribosomes; the apparent K(m) for GTP hydrolysis is 55 microM. Interestingly, in the presence of the mRNA hairpin which promotes selenocysteine incorporation, GTPase activity of SelB increases additionally by 3-4-fold; stimulation is due to kcat increasing from 0.05/min in the absence to 0.16/min in the presence of the mRNA hairpin. This mRNA-induced stimulation of SelB GTPase activity depends on the presence of ribosomes. The minimal region of the mRNA hairpin capable to stimulate GTP hydrolysis by SelB locates within the upper half of the hairpin; this part of the mRNA structure was demonstrated previously to be sufficient for binding of the mRNA to SelB. On the basis of these results, we propose that binding of the mRNA hairpin to SelB induces a conformational switch within SelB thereby promoting an increase in ribosome-mediated GTP hydrolysis.
Hüttenhofer
A
A
Lehrstuhl für Mikrobiologie, Universität München, Germany. huttenh@uni-muenster.de
Böck
A
A
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; C061878:SelB protein, Bacteria; C112705:selenocysteinyl-tRNA; D017279:Selenocysteine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases